SynBERC:MIT/Calendar/2007-7-25

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Anyone in the synthetic biology community is welcome to attend.

Wednesday July 25, 2007 at 12:30pm EST

32-D463, MIT

Anyone with questions re BioBricks or BioBricks assembly can email biobricks AT openwetware DOT org.

Topic of discussion

BioBrick™ standard biological parts "how-to" session

This session won't assume any prior knowledge regarding synthetic biology and will cover

  1. What is a BioBrick?
  2. How do I clone and assemble BioBricks?
  3. Should I use BioBrick cloning in my lab? Pro's and Cons
  4. How do I start/convert to BioBricks cloning and assembly?

If there's time, we'll have an open discussion about how to make BioBricks more generally useful to academic research labs (as opposed to just synthetic biology labs).

Outline

How do I clone and assemble BioBricks?

  • A BioBrick is a fragment of DNA with particular restriction sites upstream and downstream
  • Key enzymes and how assembly works

BioBrick Prefix

The standard BioBrick prefix depends on the part that follows it (YOUR CONNECTING PART SEQUENCE IN GREY UPPERCASE, EcoRI, NotI, XbaI, rest of the BioBrick sequence).

If the following part is a coding sequence or any other part that starts "ATG", the BioBrick prefix is:

gaattcgcggccgcttctagATG...

Otherwise, the BioBrick prefix is:

gaattcgcggccgcttctagagCA...

BioBrick Suffix

Similarly, the standard BioBrick suffix depends on the part that preceeds it. Whenever possible, the transcriptional start site of a promoter part should lie 2bp upstream of the SpeI site. (part in grey, PstI, NotI, SpeI). YOUR SEQUENCE as it should be entered into the database is in uppercase letters. For help locating the transcription initiation site in your promoter part, try promoter prediction.

The standard BioBrick suffix for most parts:

...ACtactagtagcggccgctgcag

Scars

If the second part is a CDS starting with "ATG", the scar is:

tactag

Otherwise, the scar is:

tactagag

Protein fusions

To assemble protein fusions with BioBricks, reduce the size of the scar site to 6bp for in frame protein fusions.

  • However, you must be careful not to inadvertently insert methylation sites that may decrease cutting efficiency.

The "normal scar is:

tactagag

In a protein fusion the scar is:

act aga
Thr Arg

If you use this standard, protein fusion parts cannot begin with a TC to avoid constructing a GATC methylation site that will inhibit digesting by XbaI.

Using a different frame, you may also construction fusion proteins in which the linker is Leu Asp/Glu.

Should I use BioBrick cloning?

Pros

  1. Simple, reliable (and potentially automatable) construction process
    • Restriction enzymes were chosen because they are very robust (to reaction buffers) and efficient.
    • The same reaction conditions are used every time: easier to parallelize your construction, less likely to make errors (we'll routinely be building 5-10 constructs at once).
    • Goal is to stop construction from being an experiment in itself
    • Can be used to make a construct with arbitrarily large number of components (e.g. it scales)
  2. Makes all the DNA components in your lab compatible and easily assembled.
    • No need to plan ahead ... if everything is in BioBricks form, you can easily combine components between different projects in the lab.
  3. Can track and archive parts in the Registry (if you want)
    • As long as you're happy to share the sequence and the physical DNA with others, you can get a physical backup of the DNA and archive the sequence and other information in an online database.
    • Your constructs are more reusable by others and your work can have a bigger impact.
  4. Preexisting, shared components and community of users
    • 1500 available parts including constitutive promoters, fluorescent proteins, terminators, repressors, enzymes, inducible promoters etc.
    • There is an active community sharing and documenting BioBricks (iGEM and others) that can help in debugging, recommending parts, etc (esp. here at MIT).
    • By re-using the same components across multiple groups, errors can be quickly identified and experimental caveats can be made made more obvious to new part users. In general, shared parts are more likely to be reliable than something that has only been shown to work once by one group. Making a part in an easily re-usable, sharable format immediately increases it's utility/value to the community.

Cons

  1. Leaves a mixed XbaI/SpeI sequence between any two components you assemble. The mixed sequence cannot be cut by either enzyme.
    • The added sequence can be a problem for RNA design and other sequence-specific applications
  2. You can't easily pull components out from assembled construct (except by PCR)
  3. You can't easily switch components.
    • Need to design your assembly scheme to minimize the extra assemblies necessary to change components
  4. For full compatibility, your sequence must not contain any EcoRI, XbaI, SpeI, PstI, or NotI sites.
    • You may be able to get around this in some cases
    • Why can't it have NotI sites? We don't use NotI for anything.
  5. In constructing fusion proteins, you will construct a two residues linker sequence that is either Thr Arg or Leu Asp/Glu.

Converting to BioBricks

  • Available existing BioBrick plasmids
    • Low copy and high copy, even inducible copy
    • Different antibiotic resistance markers (Amp, Tet, Cm, Kan)
  • BioBrick-compatible plasmids that you may use already
    • List existing available plasmids that can be easily made BioBricks compatible: pUC19
  • Making parts in BioBrick form
    • PCR with oligo's and tails
  • What happens if my gene has a BioBrick site in it?
    • Mutate the site via site-directed mutagenesis
    • Direct synthesis of part without site
    • You may be able to leave the sites in and still use the part

Case study

Future

  • There is work to automate this assembly process ... so if and when this comes online, you could take advantage of it.

References

Follow up

Here are some of the resources mentioned at today's lunch.

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